title: Dual fluorescence system for flow cytometric analysis of Escherichia coli transcriptional response in multi-species context.
abstract: When studying interspecies interactions in a bacterial consortium, it may be desirable to analyze one species' transcriptional response as influenced by the other species. We developed a dual fluorescence system of Escherichia coli for Fluorescence-Activated Cell Sorter (FACS)-based analysis for such a purpose. First, we generated E. coli SCC1 strain, which constitutively expresses green fluorescent protein (GFP), but otherwise showed no observable difference from the parent strain MG1655 with respect to morphology, growth, and FACS-analyzed side- and forward-scatter profiles. Next, to analyze transcriptional response, plasmids carrying promoters of interest fused to a red fluorescent protein (AsRed2) reporter, were introduced into strain SCC1. Quantification of promoter activities of araB, lacZ, fadB and rpoE via AsRed2 reporter verified that the induction levels are similar between MG1655 and SCC1 strains. In mixtures and co-cultures, GFP expression of E. coli SCC1 allowed it to be separated from non-E. coli species by FACS to purity levels of 96.7-100.0%. When a mixture of E. coli SCC1 carrying promoter-AsRed2 fusion and a non-E. coli strain was analyzed by FACS, it enabled (i) distinction of E. coli SCC1 from the non-E. coli strain, (ii) analysis of the E. coli promoter activity via AsRed2 expression and (iii) identification of transcriptional heterogeneity within the E. coli population. Co-cultures of E. coli SCC1 with Klebsiella pneumoniae and/or Enterococcus faecalis analyzed by FACS showed that E. coli fadB and rpoE transcription were differentially influenced by partner species.
authors.affiliation: School of Biological Sciences, Nanyang Technological University, Singapore.
authors.forename: Huang
authors.initials: H
authors.lastname: Miao
authors.forename: Shalini
authors.initials: S
authors.lastname: Ratnasingam
authors.forename: Chui San
authors.initials: CS
authors.lastname: Pu
authors.forename: Malan Manish
authors.initials: MM
authors.lastname: Desai
authors.forename: Chun Chau
authors.initials: CC
authors.lastname: Sze
mesh_terms: D001431:Bacteriological Techniques; D003066:Coenzyme A Ligases / Q000235:genetics; D013293:Enterococcus faecalis; D004926:Escherichia coli / Q000166:cytology* / Q000235:genetics*; D029968:Escherichia coli Proteins / Q000235:genetics; D005434:Flow Cytometry / Q000379:methods*; D015964:Gene Expression Regulation, Bacterial / Q000502:physiology*; D017930:Genes, Reporter; D018740:Genetic Heterogeneity; D049452:Green Fluorescent Proteins; D007711:Klebsiella pneumoniae; D049408:Luminescent Agents; D008164:Luminescent Proteins; D011401:Promoter Regions, Genetic; D011993:Recombinant Fusion Proteins; D012808:Sigma Factor / Q000235:genetics; D013045:Species Specificity; D015533:Transcriptional Activation / Q000502:physiology*; D000097573:Red Fluorescent Protein
chemical_list: D003066:Coenzyme A Ligases; D029968:Escherichia coli Proteins; D049452:Green Fluorescent Proteins; D049408:Luminescent Agents; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D012808:Sigma Factor; D000097573:Red Fluorescent Protein; C063202:sporulation-specific sigma factors; C085934:acyl-coenzyme A synthetase, E coli